Development of NIRS method for quality control of drug combination artesunate-azithromycin for the treatment of severe malaria.

Near infrared spectroscopy (NIRS) methods were developed for the determination of analytical content of an antimalarial-antibiotic (artesunate and azithromycin) co-formulation in hard gelatin capsule (HGC). The NIRS consists of pre-processing treatment of spectra (raw spectra and first-derivation of two spectral zones), a unique principal component analysis model to ensure the specificity and then two partial least-squares regression models for the determination content of each active pharmaceutical ingredient. The NIRS methods were developed and validated with no reference method, since the manufacturing process of HGC is basically mixed excipients with active pharmaceutical ingredients. The accuracy profiles showed ß-expectation tolerance limits within the acceptance limits (±5%). The analytical control approach performed by reversed phase (HPLC) required two different methods involving two different preparation and chromatographic methods. NIRS offers advantages in terms of lower costs of equipment and procedures, time saving, environmentally friendly.

Investigation of porous graphitic carbon at high-temperature liquid chromatography with evaporative light scattering detection for the analysis of the drug combination artesunate--azithromycin for the treatment of severe malaria.

Artesunate combined therapies represent the best option for the treatment of malaria and require the development of new methods of analysis. Retention, selectivity and detection with high-temperature liquid chromatography-porous graphitic carbon-evaporative light scattering detection was studied for artesunate and azithromycin separation. Organic solvent, concentration of organic modifiers, temperature and flow rate were all relevant parameters to optimize this separation. The behaviour of artesunate in the tested conditions appeared close to a neutral compound. In CH(3)OH, only azithromycin retention was dramatically altered depending on the [triethylamine]/[formic acid] ratio and on the temperature, whereas in CH(3)CN, azithromycin, artesunate, artemisinin and dihydroartemisinin retentions decreased with the temperature increase whatever the organic modifier ratio. The best efficiency was obtained with CH(3)CN. 25% variation of the concentration values of the organic modifiers did not significantly influenced the retention. The sensitivity of ELSD increased with the flow rate decrease. Peak area and S/N ratio dramatically decreased with the flow rate increase by 10- and 5-fold for artesunate and azithromycin, respectively. Non-linear calibration curves were obtained for both artesunate and azithromycin.

Development and validation of a rapid capillary electrophoresis method for the determination of oseltamivir phosphate in Tamiflu and generic versions.

A rapid and reliable capillary zone electrophoresis method was developed and validated for the assay of oseltamivir phosphate in capsules. Separation was carried out in fused silica capillary (60.2 cm total length and 10.0 cm effective length, 75 microm i.d.) by applying a potential of -15 kV at 25 degrees C. The selected electrophoretic buffer consisted of 50 mM sodium phosphate, pH 6.3 (direct UV detection, 226 nm). A short electrophoretic analysis time (less than 1.5 min) was obtained using the short end injection mode. The method was validated in terms of specificity, linearity, precision and accuracy. The RSD values were 0.94 and 0.98% for repeatability and intermediate precision, respectively. Recovery determinations allowed the calculation of a confidence interval from 98.64 to 100.26% with a relative standard deviation value of 0.38%. LOD and LOQ were estimated at 0.97 and 3.24 microg/mL, respectively. The validated method was successfully applied to the determination of oseltamivir in three commercially available capsules (Tamiflu, Saiflu and Flufy). The results were in good agreement with those obtained by a HPLC method previously developed in our laboratory. This method presents advantages including short run time, simple and rapid sample preparation and no use of non-aqueous solvent throughout the analysis.

Determination of artesunate using reversed-phase HPLC at increased temperature and ELSD detection.

Artesunate (ART) determination can be performed by evaporative light scattering detection with mobile phase composed of CH(3)CN/HCOOH 0.01 M (40:60 v/v; pH 2.85). Evaporative light scattering detection instead of UV detection allowed to improve the sensitivity and the LOD. However, the evaporative light scattering detection response of dihydro-artemisinin appears weaker than for ART, whereas with UV detection the response of ART and dihydroartemisinin seemed similar. Constant analysis time was obtained on using the mobile phase with a flow rate of 0.5 mL/min and column temperature at 60 degrees C instead of 0.7 mL/min at room temperature. This led to less solvent consumption. Moreover, decrease in the flow rate and increase in the column temperature were advantageous for higher sensitivity with both evaporative light scattering detection and UV detection. ART determination in rectal gel and suppositories were compared with these different detection modes and similar results were obtained.

In vitro release and stability of an artesunate rectal gel suitable for pediatric use.

The rectal route is indicated to treat patients with rapidly evolving malaria who cannot take oral medication to prevent progression to severe forms of the disease. Improvement can be made in terms of rectal bioavailability and stability of current formulations. We studied a new two-compartment, muco-adhesive gel formulation of artesunate which is adapted for use in children and storage in tropical climates. The formulation contains 50mg of artesunate per gram of gel. Because of its instability in aqueous solutions, artesunate is in the dry component of the gel with Carbopol and separate from the liquid phase until reconstitution. Artesunate is stable in the dry blend for 6 months at 45 degrees C and 60% RH. The gel should be used between 1 and 72 h after being reconstituted. Artesunate release was measured by with a rapid, simple and reliable HPLC-UV which allowed the analysis of artesunate and dihydroartemisinin with an analysis time at 3 min. The amount of artesunate released over 6h was 56 +/- 0.97%. Compared to the reference suspension, total release and dissolution efficiency were lower and rate of release was slower (time to 50% dissolution 271 +/- 21 min), probably because of the higher viscosity of the gel, but the drug release profiles were similar. The calculated in vitro release exponent (n) value suggested that artesunate is released from the gel by non-Fickian transport.

Determination of N,N'-ethylenebisstearamide additive in polymer by normal phase liquid chromatography with evaporative light scattering detection.

A new method for N,N'-ethylenebisstearamide (EBS) analysis was developed and validated in normal phase-HP liquid chromatography (NP-HPLC) with diol column at 50 degrees C with 100% CHCl(3) at 1 mL min(-1) and evaporative light scattering detection with elution time at 3.0 min. EBS solubility was the best at 0.80 gL(-1) in CHCl(3)/methanol 90:10. The molecular structure of commercial samples of EBS was determined by GC-MS which ascertained that the main structure is C18/C18 at approximately 45%. The remaining part was constituted by molecules with different alkyl chain length. The HPLC quantification method was proved linear (r=0.9983), accurate (99.6%) and precise (1.95%). Limit of quantification (LOQ) and limit of detection (LOD) were equal to 2.0 and 0.8 microg mL(-1), respectively. The suitability of this method was assessed with a dissolution/precipitation extraction procedure of EBS from ethylene vinyl acetate (EVA) polymer which showed that other additives and polymer do not interfere with EBS analysis. The intra-day and day-to-day precisions of extraction method were equal to 9.1% and 9.9%, respectively.

Stability of artesunate in pharmaceutical solvents.

Stability of artesunate (ART) was established in three pharmaceutical solvents. The chromatographic conditions developed for this study were acetonitrile:potassium phosphate buffer 10 mM (40:60, v:v; pH 2.9) at 0.7 mL min(-1) with UV detection at 220 nm using a short X-Terra RP C18 column (50 mm x 3 mm, 3.5 microm). This isocratic condition led to the separation between ART and its main degradation products (i.e. alpha-DHA and beta-DHA) with analysis time of less than 4 min. The retention factors are 1.49, 2.26 and 2.79 min for alpha-DHA, beta-DHA and ART, respectively. This method was proved linear (r(2)=0.9995), accurate (R.S.D.=0.20), precise (R.S.D.=0.74) and robust. The system performance remained unaffected by pH variation from 2.6 to 3.2 and variation of acetonitrile percentage from 38 to 42. Stability of ART was assessed in ethanol, propylene glycol (PG) and polyethylene glycol 400 (PEG 400). Unfortunately none of these solvents prevented ART from degradation longer than 3 months. In ethanol, significant degradation of ART occurred after 3 months at room temperature and this degradation was characterised by numerous degradation products. In PEG 400, significant degradation was observed after only 1 month, however DHA was the unique degradation product, which is also an efficient anti-malarial drug.

Development and validation of a capillary electrophoresis method for the determination of sulfate in effervescent tablets.

A suitable capillary electrophoresis (CE) method was developed and validated for sulfate anion determination in effervescent tablets of Digedryl. The large excess of other ions in the matrix (i.e. excipients) constituted the main difficulty of this method's development. So an original analytical procedure for both the conditioning and rinsing of the capillary was purposed including a running electrolyte constituted by boric acid 20 mM and hexamethonium dibromide 0.75 mM at pH 8.00. Separation was carried out on a 60.2 cm (50 cm to the detector) x 0.75 microm i.d. fused-silica capillary at a potential of -29 kV and 35 degrees C. Indirect UV detection was performed at a wavelength of 254 nm using a background electrolyte containing potassium chromate. Nitrate anion was used as an internal standard for quantification. This CE method was validated in terms of selectivity, linearity, accuracy and precision.

Molecular lipophilicity determination of a huperzine series by HPLC: comparison of C18 and IAM stationary phases.

Two hydrophobic parameters (logkw-C18 and logkw-IAM, respectively) of a huperzine A series were extrapolated by high performance liquid chromatography (HPLC) using both C18 and immobilised artificial membrane (IAM) columns. A mathematical correlation between C18 and IAM hydrophobic parameters was completed, suggesting a similar behaviour on both columns. This behaviour was principally led by hydrophobic forces. The theoretical lipophilicity (logP) of each compound was computed using Pallas software and compared to experimental values, showing a similar lipophilic behaviour. Finally, the huperzine logkw-IAM and logkw-C18 values were correlated with the relative bound percentage of huperzine in human serum albumin, confirming that hydrophobic forces are predominant in the huperzine-HSA binding mechanism.

Protonation equilibrium and lipophilicity of moxifloxacin.

This study was performed to characterise the protonation equilibrium at the molecular level and pH-dependent lipophilicity of moxifloxacin. After determining macro- and micro-constants, distribution features of four microspecies in aqueous phase were assessed. The apparent partition coefficient versus pH profile of moxifloxacin showed a parabolic curve in n-octanol/buffer system which reached near pI. The true partition coefficient was calculated from the log P(app) and microconstants values.